CN102428184A - Marker for prognosis of liver cancer - Google Patents

Marker for prognosis of liver cancer Download PDF

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CN102428184A
CN102428184A CN2010800213900A CN201080021390A CN102428184A CN 102428184 A CN102428184 A CN 102428184A CN 2010800213900 A CN2010800213900 A CN 2010800213900A CN 201080021390 A CN201080021390 A CN 201080021390A CN 102428184 A CN102428184 A CN 102428184A
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prognosis
hcc
ncbi
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affinity tag
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朴真永
洪锡珠
金种旻
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Shen Yingze
CBS Bioscience Co Ltd
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Shen Yingze
CBS Bioscience Co Ltd
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    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57438—Specifically defined cancers of liver, pancreas or kidney
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    • G01—MEASURING; TESTING
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers
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Abstract

The present invention relates to: a marker for the prognosis of liver cancer; a composition for estimating the prognosis of liver cancer, which contains a substance for detecting a change in the expression level of the prognostic marker for liver cancer; a kit for estimating the prognosis of liver cancer, which contains the composition for estimating liver cancer prognosis; a method for estimating the prognosis of liver cancer using the marker for liver cancer prognosis; and a method for screening a therapeutic agent for liver cancer using the marker for the prognosis of liver cancer.

Description

The prognosis in hcc affinity tag
Technical field
The present invention relates to prognosis in hcc affinity tag (marker for prognosis of liver cancer); Relate to the compsn that is used to assess prognosis in hcc, said composition comprises the material of the changes of expression level that can detect said prognosis in hcc affinity tag; Relate to the test kit that is used to assess prognosis in hcc, this test kit comprises the said compsn that is used to assess prognosis in hcc; Relate to the method for using said prognosis in hcc affinity tag to carry out the prognosis in hcc assessment; And relate to the method for using said prognosis in hcc affinity tag to screen the therapeutical agent of liver cancer.
Background technology
Cancer has identical meaning with malignant tumour.It is meant a kind of like this disease, because a variety of causes, the function of regulating cell proliferation suffers damage, thereby therefore unusual cell is uncontrolled and hyper-proliferative penetrates into tissue and organ on every side, forms block and destroys normal organizing.Because the quick growth of cancer, infiltrative (penetrance or diffusion) growth, diffusion reasons such as (moving far from its generation area), the cancer that comes from various tissues in the human body bring danger can for people's life.
In the middle of cancer, known liver cancer is one of the most fatal in the world cancer.Especially, claim according to reports, the African Realm in the Asia with time the Sahara, annual at least about there being 500,000 people to die from liver cancer.Liver cancer mainly is divided into the hepatocellular carcinoma that originates from liver cell self and cancer are diffused to liver by its hetero-organization secondary liver cancer.Approximately at least 90% liver cancer is hepatocellular carcinoma, and common sense term liver cancer is meant hepatocellular carcinoma.
The cause of most of liver cancer is the acute or chronic infections that receive hepatitis B virus or hepatitis C virus according to reports.Yet, about the intracellular molecular mechanism of liver cancer generation and development is not also illustrated.According to tradition research; Claim according to reports under the situation of over-expresses or excessive activation being mutated into oncogene owing to a variety of causes such as proto-oncogenes such as various growth factor genes; Perhaps hang down under the situation of expression or loss of function owing to a variety of causes suddenlys change, can cause the generation and the development of the multiple cancer that comprises liver cancer at the tumor suppressor gene such as Rb albumen or p53 albumen.Especially, about liver cancer, genes such as the verified for example p53 through modification, beta-catenin white (beta-catenin), AXINI, p21 (WAF1/CIP1) and p27Kip are relevant with it.But, recognize that recently generation and the development of most of cancers that comprises liver cancer be because independent specific gene, but since the complicacy of the several genes relevant with cell cycle, signal transmission etc. interact.Thereby, be necessary from only concentrate on discrete gene or proteic expression or function, to spin off, and carry out range gene or proteic holistic approach.
The prognosis of liver cancer is meant that prediction suffers from patient's the various situation of liver cancer, the possibility of rehabilitation comprehensively from liver cancer for example, the possibility that recurs through the treatment back, possibility that the patient survives behind the diagnosing cancer of liver or the like.This can change according to various situation such as severity of disease, Diagnostic Time, therapeutic advances.Have only when suitably using various treat-ment according to its prognosis, liver cancer can obtain the effective treatment.For example,, be necessary to avoid causing the treat-ment of the danger of serious side effects to the patient for being assessed as the patient that good prognosis is arranged, for example radical chemotherapy or operation, radiotherapy etc., and select those gentle relatively, conservative and safe treat-ment.On the other hand, for being assessed as the patient that relatively poor prognosis is arranged, should carry out chemotherapy or operation energetically, for example treat-ment such as radiotherapy is to try hard to increase lifetime or survival rate.
According to the research of carrying out up to now, the prognosis of the liver cancer that has developed is very bad, has shown that self diagnosis plays high lethality rate dead in 6 months, and residue is four months average duration of life only.But size has good prognosis less than 3 centimetres liver cancer, and knownly need not any special treatment, and the survival rate in 1 year is 90%, and the back 5 years survival rate of performing the operation is about 40～50%.Yet, be difficult to accurately assess the prognosis of liver cancer patient according to prior art.For the accurate assessment prognosis, need a kind of analytical procedure that is divided into the patient each risk cohort.Yet up to now, the clinical pathology liver cancer stage when only relying on diagnosis and first operative treatment is confirmed prognosis, and does not have a kind of method that is used for accurate assessment liver cancer.But, unfortunately, only can't accurately confirm the prognosis of each liver cancer patient through the liver cancer stage.
CBS albumen (cystathionine beta-synthase) is a kind of enzyme that homocysteine is transformed into cystathionine; Known its works in changeing sulphur (transsulfuration) path, and [the J Biol Chem.1994 May 20 that plays a significant role in the methionine(Met) metabolism in vivo; 269 (20): 14835-40].
NNMT albumen (vitamin PP N methyltransgerase) is a kind of methylated enzyme of N-that carries out vitamin PP and pyridine, relevant [the Genomics.1998 Sep 15 of known its metabolism with various medicines and heteroplasia thing; 52 (3): 312-24].
TKT albumen (transketolase) be a kind of in non-oxide phosphopentose pathway the enzyme of performance central role, as the member of this family, known have TKTL1 (change keto-alcohol appearance enzyme 1), TKTL2 (changeing keto-alcohol appearance enzyme 2) [J Biol Chem.1993 Jan 15; 268 (2): 1397-404].
AIFM1 albumen (apoptosis inducing factor 1 that plastosome is relevant) is the flavoprotein that is also referred to as AIF.Known if apoptosis takes place, then this albumen moves to nucleus, causes chromosomal gathering and fracture, and induces secretion [the Nature.1999 Feb 4 from mitochondrial cytochrome c and caspase 9; 397 (6718): 441-6].
AKT albumen is and AKT2, AKT3 proteins associated; And known its transmitting (promptly through PI 3 kinases growth factor signal; The signal transmission of platelet derived growth factor (PDGF), Urogastron (EGF), Regular Insulin and insulin-like growth factor I (IGF-I) etc.) works in; When it is activated, known can the phosphorylation apoptosis-related protein and make their inactivations [Proc Natl Acad Sci U S is Jul A.1987; 84 (14): 5034-7].
ATG3 albumen (autophagy be correlated with 3 homologues) was participated in such as combining between people ATG8 homologues such as GATE-16, GABARAP, MAP-LC3 and the lipid.Have and report that the mouse that lacks ATG3 does not form cytolysosome, and in birth one day after death [J Biol Chem.2002 Apr 19; 277 (16): 13739-44.Epub 2002 Feb 1].
ATG5 albumen (autophagy be correlated with 5 homologues) is relevant with autophagocytosis.ATG5 is known can be through the effect formation ATG12-ATG5 conjugate of ATG7 and ATG10, and this ATG12-ATG5 conjugate moves to the cytolysosome film and sentences in the combination of ATG8 and phosphatidylethanolamine and work.Have and report that the mouse that lacks ATG5 does not form cytolysosome, and dead immediately after birth, and secretion and the interior caspase 9 [Nature 432:1032-1036 (2004)] of activation of mitochondria that the ATG5 that blocks can inducing cell pigment c.
ATG7 albumen (autophagy be correlated with 7 homologues) is relevant with autophagocytosis.Have report to claim that ATG7 forms the ATG12-ATG5 conjugate with ATG10, and ATG7 participate in the combination of ATG8 and phosphatidylethanolamine with ATG3.The mouse that lacks ATG7 does not form cytolysosome, and after birth dead immediately [J Biol Chem.2001 Jan 19; 276 (3): 1701-6.Epub 2000 Nov 28].
ATG12 albumen (autophagy be correlated with 12 homologues) is relevant with autophagocytosis.Known ATG12 is through the effect formation ATG12-ATG5 conjugate of ATG7 and ATG10, and this ATG12-ATG5 conjugate moves to the cytolysosome film and sentences in the combination of ATG8 and phosphatidylethanolamine [the J Biol Chem.1998 Dec 18 that works; 273 (51): 33889-92].
A member of BAX albumen (BCL2 be correlated with X protein) BCL2 albumen family, known its through with BCL2 combine to promote apoptosis, and with the loss of mitochondrial membrane potential and relevant [the Cell.1993 Aug 27 of secretion of cytochrome c; 74 (4): 609-19].
BCL2 albumen (B cell lymphoma albumen 2) is a kind of mitochondrial membranin, can suppress apoptosis.Known its suppresses cytochrome c from mitochondrial secretion, activate caspase and through with the apoptosis incitant combine suppress apoptosis [Proc Natl Acad Sci U S is Jul A.1986; 83 (14): 5214-8].
BCL2L1 albumen (Bcl-2 appearance 1 albumen) is a member of BCL2 protein groups, and known its is positioned at mitochondrial adventitia and relevant with the distribution of mitochondrial membrane passage.It exists with two kinds of hypotypes.The BCL-XL of more microscler formula is known can to suppress apoptosis, and can promote apoptosis [Cell.1993 Aug 27 than the BCL-XS of short-form; 74 (4): 597-608].
BNIP3 albumen (BCL2/ adenovirus E 1 B 19kD-interaction protein 3) can combine with E1B 19kDa albumen and BCL2, and known can cell death inducing and autophagocytosis [J Exp Med.1997 Dec 15; 186 (12): 1975-83].
CASP8 albumen (caspase 8, the caspase that apoptosis is relevant) is the enzyme that belongs to caspase family, and it is at acting enzyme of Apoptosis Mechanism starting stage through FAS.If its through with FADD combine activate with the cutting of proteolyze property, its activates various other caspases, and [Proc Natl Acad Sci U S is Dec 10 A.1996; 93 (25): 14486-91; Biochem is Aug 15 J.1997; 326 (Pt 1): 1-16].
CSE1L albumen (a kind of CSE1 chromosome segregation) is known to work in that importin-α is sent to the tenuigenin from nucleus once more, and relevant with apoptosis or cell proliferation [Proc Natl Acad Sci U S is Oct 24 A.1995; 92 (22): 10427-31].
DIABLO albumen (the direct IAP with low iso-electric point is conjugated protein) is a kind of Intramitochondrial albumen that is positioned at; And if apoptosis took place, then it would move to tenuigenin and combines activation [the Cell.2000 Jul 7 with the help caspase with IAP (apoptosis inhibitor albumen); 102 (1): 43-53].
DRAM albumen (damage regulate autophagy modulin) is a kind of lysosomal membranin, and known its autophagocytosis with the control of p53 tumor suppression is relevant and in the apoptosis that p53 controls, be necessary [Cell.2006 Jul 14; 126 (1): 121-34].
E2F1 albumen (E2F transcription factor 1) is a member of E2F family transcription factor; The control of known itself and cell cycle and tumor suppression is closely related, and through promoting the cell growth or induce the apoptosis relevant with p53 [Gene.1996 Sep 16 with the proteic combination of Rb; 173 (2): 163-9].
(APO-1, CD95 TNFRSF6) are a member of TNF receptor family to FAS albumen.Known its accepted the signal of FAS part and formed DISC (dead inducement signal mixture) with FADD (Fas associated death domain protein), caspase 8 and caspase 10 and come cell death inducing [J Biol Chem.1992 May 25; 267 (15): 10709-15].
FRAP1 albumen (Mammals rapamycin target protein) is also referred to as mTOR.Known its regulated such as pressure-dependent reactions such as dna damage, nutrition in the cell exhaust, and to comprise the TORC2 mixture of FRAP1 known be target spot [the Nature.1994 Jun 30 of the inhibition immunization of cell-cycle arrest and FKBP12-rapamycin mixture; 369 (6483): 756-8].
LAMP1 albumen (lysosome related membrane protein 1) is also referred to as CD107a antigen.As if it is a kind of membrane glycoprotein, and relevant [J Biol Chem.1990 May 5 with the diffusion of cancer cells; 265 (13): 7548-51].
LC3 albumen (3 kinds of albumen 1 of MAP1 light chain) is microtubule bindin, and it is relevant with the interaction between microtubule and cytoskeleton.LC3 albumen is the homologue of yeast ATG8, and known its can combine and form cytolysosome [J Biol Chem.1994 Apr 15 through the proteic effect of multiple autophagy with phosphatidylethanolamine; 269 (15): 11492-7].
PRKAA1 albumen (AMP activated protein kinase, catalytic, α-1) is the catalytic subunit of AMPK.This AMPK is the albumen that plays the effect that detects the intracellular energy level.Known it when the AMP/ATP ratio increases, activating to limit various biosynthesizing reaction [FEBS Lett.1994 Dec 12; 356 (1): 117-21].
Pten protein (Phosphoric acid esterase and tensin homologue) is known to be a kind of tumor suppression, inactivation in polytype cancer.It is a kind of phosphoprotein phosphatase also be a kind of PI-3,4 simultaneously, 5-triphosphoric acid 3 Phosphoric acid esterases, known its can destroy the PI3K-AKT/PKB signal and transmit [Nat Genet.1997 Apr; 15 (4): 356-62].
ULK1 albumen (Unc-51 appearance kinases 1) is a kind of serine/threonine kinase relevant with axon growth, is also referred to as the ATG1 homologue.For autophagocytosis, known its can be by mTOR phosphorylation [Genomics.1998 Jul 1; 51 (1): 76-85].
XIAP albumen (the chain apoptotic proteins of X suppresses son) is a member of IAP family, and in IAP, it has strong apoptosis to suppress effect.Known its through with activity [the Nature.1996 Jan 25 that suppresses apoptosis and suppress caspase of combining of Tumor Necrosis Factor Receptors correlation factor TRAF1, TRAF2; 379 (6563): 349-53].
But how not clear these protein expression levels and expression pattern specifically change in liver organization, and how to use these albumen assessment prognosis in hcc.And the gene that does not also use albumen or proteins encoded up to now is as the instance that is used for the prognosis in hcc affinity tag.
Summary of the invention
Technical task
An object of the present invention is to provide a kind of biomarker of relevant prognosis in hcc, to assess the prognosis of liver cancer patient easily and accurately and also to provide an exploitation to be used for the important starting point of the therapeutical agent of liver cancer.Thereby, the invention provides a kind of prognosis in hcc affinity tag; A kind of compsn that is used to assess prognosis in hcc, said composition comprises the material of the changes of expression level that can detect said prognosis in hcc affinity tag; A kind of test kit that is used to assess prognosis in hcc, said test kit comprise the said compsn that is used to assess prognosis in hcc; A kind of method of using said prognosis in hcc affinity tag to carry out the prognosis in hcc assessment; And a kind ofly use said prognosis in hcc affinity tag to screen the method for the therapeutical agent of liver cancer.
Technical scheme
The present invention relatively takes from the degree of gene expression in the liver cancer tissue that multidigit is diagnosed as the patient who suffers from liver cancer; And according to every patient's progress detection specificity gene a large amount of or that express in a small amount, thereby find can be used in the prognosis in hcc affinity tag of assessing prognosis in hcc.
First aspect of the present invention relates to the prognosis in hcc affinity tag, and said prognosis in hcc affinity tag comprises and is selected from by one in the group of following genomic constitution or the combination of at least two genes:
CBS (cystathionine beta-synthase; NCBI GI:209862802; SEQ ID NO:79);
NNMT (NNMT; NCBI GI:62953139; SEQ ID NO:80);
TKT (transketolase; NCBI GI:205277461; SEQ ID NO:81);
AIFM1 (the apoptosis inducing factor 1 that plastosome is relevant; NCBI GI:22202627; SEQ ID NO:82);
AKT1 (RAC-α serine/threonine protein kitase; NCBI GI:62241010; SEQ ID NO:83);
ATG3 (autophagy GAP-associated protein GAP 3; NCBI GI:34147490; SEQ ID NO:84);
ATG5 (autophagy GAP-associated protein GAP 5; NCBI GI:92859692; SEQ ID NO:85);
ATG7 (autophagy GAP-associated protein GAP 7; NCBI GI:222144225; SEQ ID NO:86);
ATG12 (autophagy correlated protein 12; NCBI GI:38261968; SEQ ID NO:87);
BAX (apoptosis regulatory factor BAX; NCBI GI:34335114; SEQ ID NO:88);
BCL2 (apoptosis regulatory factor Bcl-2; NCBI GI:72198188; SEQ ID NO:89);
BCL2L1 (apoptosis regulatory factor Bcl-X; NCBI GI:20336333; SEQ ID NO:90);
BNIP3 (BCL2/ adenovirus E 1 B 19kD interaction protein 3; NCBI GI:7669480; SEQ ID NO:91);
CASP8 (caspase 8; NCBI GI:122056470; SEQ ID NO:92);
CSE1L(Exportin-2；NCBI?GI：29029558；SEQ?ID?NO：93)；
DIABLO (Diablo homologue, plastosome; NCBI GI:218505810; SEQ ID NO:94);
(the autophagy modulin is regulated in damage to DRAM; NCBI GI:110825977; SEQ ID NO:95);
E2F1 (transcription factor E2F1; NCBI GI:168480109; SEQ ID NO:96);
FAS (tumor necrosis factor receptor super family member 6; NCBI GI:23510419; SEQ ID NO:97);
FRAP1 (FKBP12-Wyeth-Ayerst Laboratories compound relative protein; NCBI GI:206725550; SEQ ID NO:98);
LAMP1 (lysosome related film gp 1; NCBI GI:112380627; SEQ ID NO:99);
LC3 [MAP1LC3A] (microtubule bindin 1A/1B light chain 3A; NCBI GI:31563519; SEQ ID NO:100);
PRKAA1 (5 '-AMP activated protein kinase catalytic subunit α-1; NCBI GI:94557300; SEQ ID NO:101);
PTEN (PI-3,4, the phosphoprotein phosphatase PTEN of 5-triphosphoric acid 3 Phosphoric acid esterases and dual specific; NCBI GI:110224474; SEQ ID NO:102);
ULK1 (serine/threonine protein kitase ULK1; NCBI GI:225637564; SEQ ID NO:103); With
XIAP (the albumen 4 that contains baculovirus IAP Tumor-necrosis factor glycoproteins; NCBI GI:32528298; SEQ ID NO:104).
The prognosis of liver cancer can be assessed from many aspects.Yet, typically confirm from possibility, the possibility of existence and the aspects such as possibility of DFS of recurrence.Research described in this specification sheets is carried out through following manner: from the liver cancer tissue of multidigit liver cancer patient, obtain gene expression profile; The possibility of the overall possibility of considering recurrence, existence and the aspects such as possibility of DFS to be detecting the gene in the expression level significant difference through carrying out the statistic data treating processes, and find to assess the affinity tag of prognosis in hcc.
In this manual, " prognosis in hcc affinity tag " is meant a kind of gene marker, and it is to be used to assess liver cancer the standard that the back patient has prognosis good or difference takes place.Among the present invention, in the patient's that good prognosis is arranged liver cancer tissue the expression level of this affinity tag with compare high or low significantly through testing predetermined standard level.Especially, in the present invention, this affinity tag has significantly low p value, and this value is based on differential expression calculating in patient's the liver cancer tissue of good prognosis and relatively poor prognosis.Preferably, the p value is less than 0.05.
For the known liver cancer tissue of suffering from the patient of liver cancer of multidigit, the gene expression profile and the standard level comparison of the patient of the present invention through good or relatively poor therapeutic advance will be arranged liver cancer tissue are analyzed.Therefore, the affinity tag of so confirming can the specificity difference has the patient of good prognosis in hcc or relatively poor prognosis in hcc, thereby it can be used for the assessment of prognosis in hcc effectively.And; Consider specificity great expression or expression in a small amount in the liver cancer tissue of affinity tag in the patient of specific prognosis is arranged of so confirming; The physiologic function of this affinity tag might directly relate to the prognosis of the generation of relevant liver cancer and the physiologic function of progress, especially liver cancer.Therefore, this affinity tag can be used as the target in the research of generation and progress of liver cancer effectively or be used for the exploitation of the therapeutical agent of liver cancer.
Especially, the statistical study that is based on unprecedented multidigit patient's liver cancer tissue at the prognosis in hcc affinity tag in aspect first of the present invention is found.Thereby; With based on the gene expression profile of some patients' liver cancer tissue only and the conventional affinity tag of finding about liver cancer compare; Said affinity tag has bigger significance, has higher clinical utility value, and has the power of assessing more accurately that is used to assess prognosis in hcc.
Second aspect of the present invention relates to the compsn that is used to assess prognosis in hcc, and said compsn includes the expression level of the prognosis in hcc affinity tag described in can the specific detection first aspect and/or the material of expression pattern.
Here, can be through confirming to detect the expression level or the expression pattern of every kind of prognosis in hcc affinity tag by the general biochemical analysis method of level or pattern that corresponding gene is transcribed the mRNA of generation.Be used for like this confirming that the level of mRNA or the analytical procedure of pattern have RT-PCR, competitive RT-PCR, real-time RT-PCR, the experiment of RNA enzyme protection, the Northern marking, dna microarray or the like.In addition, can use normally used method in association area.
Through above-mentioned analytical procedure, can the level of the mRNA in the biological sample of liver cancer patient or pattern and standard level be compared, and therefrom detect the difference of the expression level and/or the expression pattern of prognosis in hcc affinity tag of the present invention.This makes it possible to assess the prognosis of liver cancer patient.In this manual; " biological sample " is meant the expression level or the expression pattern that can detect said prognosis in hcc affinity tag, and the separation that perhaps has level by coded proteic of said prognosis in hcc affinity tag or have a pattern is from human inner cell, tissue or the like.It can for example be urine, blood, blood plasma, serum etc., but does not specifically only limit to these.
Be used for including the special primer of the mRNA of said prognosis in hcc affinity tag of the present invention through the test kit that RT-PCR measures level or the pattern of mRNA.In this manual, " primer " is meant and can complementally combines and make reversed transcriptive enzyme or archaeal dna polymerase can start the nucleotide sequence with free 3 ' hydroxyl of template duplicating with template.Primer is the Nucleotide that has with the sequence of specific gene nucleic acid array complementation, can use the about 7bp～50bp of length, the preferred primer of about 10bp～30bp.Other RT-PCR test kit can comprise container, reaction buffer, deoxynucleotide (dNTP), enzyme such as Taq polysaccharase that test tube or other are fit to and reversed transcriptive enzyme, DNA enzyme, RNA enzyme inhibitors, DEPC-water, sterilized water etc. according to concrete embodiment.Under suitable buffered soln neutral temperature, primer can start the synthetic of DNA under the situation of polymerization agent (being archaeal dna polymerase or reversed transcriptive enzyme) and four kinds of different nucleoside triphosphates and temperature existence.Said primer can comprise other base sequences that do not change at the essential property of the synthetic acting primer of starting point of DNA.Can use known method chemosynthesis primer, and can use that known several different methods transforms nucleotide sequence in the association area.
" expression level that can specific detection prognosis in hcc affinity tag and/or the material of expression pattern " in second aspect of the present invention can be any material that specificity is used for the respective markers thing in the analytical procedure of the expression level of corresponding prognosis in hcc affinity tag or expression pattern; Or at least two kinds combination in the said material, be not limited to be used for the primer of RT-PCR.The technical characteristic of second aspect of the present invention is the expression level of the said prognosis in hcc affinity tag in the liver cancer tissue that is tried liver cancer patient or expression pattern and standard level comparison, and detects difference.Thereby any material that can detect such difference can be used as " expression level that can specific detection prognosis in hcc affinity tag and/or the material of expression pattern ", and realizes object technology effect of the present invention.And those of ordinary skill in the art can select and use suitable material according to concrete embodiment with reference to the common practise of association area.
The 3rd aspect of the present invention relates to the compsn that is used to assess prognosis in hcc, and said compsn includes the coded proteic material that has level and/or have pattern of said prognosis in hcc affinity tag in can the specific detection first aspect.
Expression level or expression pattern about said prognosis in hcc affinity tag of the present invention; The method of detection in second aspect of the present invention according to the level of the mRNA of each affinity tag or pattern, can also use to be used for detecting coded proteic of each affinity tag of sample and to have level or exist pattern with the expression level of estimating each affinity tag or the method for expression pattern.
There is level in coded proteic of the affinity tag of prognosis in hcc described in the present invention or exists the specific detection of pattern to be meant confirms the albumen and its method that exists with which kind of pattern that exist prognosis in hcc affinity tag described in how many present invention coded in the biological sample.For example, be enough in the antibody capable of said prognosis in hcc affinity tag encoded protein specific combination and confirm the process that there is level or has pattern.In this specification sheets, " antibody " be meant can with the albumen of antigenic epi-position specific combination, it is a notion that includes polyclonal antibody, monoclonal antibody and recombinant antibodies.
For using the proteic method that has level or have pattern of TPPA, western blotting, ELISA (enzyme-linked immunosorbent assay), radioimmunoassay, radioimmunodiffusion, Ouchterlony immunodiffusion(ID), rocket immunoelectrophoresis (rocket immunoelectrophoresis), histogenic immunity dyeing, immune precipitation determination, complement fixation test (CFT), FACS (fluorescent activation cell divide), protein chip etc. are arranged.But, except that above-mentioned, can use any method that in association area, generally adopts.
Through above-mentioned analytical procedure; Can be with formation level or the formation pattern and the standard level comparison of the immune complex in the biological sample that is tried liver cancer patient; And can confirm therefrom that there is level in coded proteic of said prognosis in hcc affinity tag of the present invention or has pattern, and finally can assess the prognosis of liver cancer patient.
Here, " immune complex " is meant the mixture of coded albumen of said prognosis in hcc affinity tag and its specific antibody.Generally through detecting and the size of two anti-link coupled certification mark signals and formation level or the formation pattern that pattern is measured immune complex.Such certification mark for example can be enzyme, fluorescent substance, part, luminophore, nanoparticle, redox molecule, ri or the like, but is not limited to these.When using enzyme as certification mark; As the enzyme that can use β-glucuronidase (β-gluculonidase), β-D-glucuroide, beta-D-galactosidase, urease, px or SEAP, E.C. 3.1.1.7, P-FAD, HK and GDP enzyme, rnase, P-FAD, luciferase, phosphofructokinase, phosphoric acid enol pyruvic acid carboxylase, SGOT, PEP decarboxylase, β-Nei Xiananmei or the like, but said enzyme is not limited to these are arranged.When using fluorescent substance as certification mark; As the fluorescent substance that can use resorcinolphthalein (fluorescein), isothiocyanate, rhodamine, phycoerythrobilin, phycocyanin, allophycocyanin, OPA, fluorescamine etc. are arranged, but said fluorescent substance is not limited to these.When using part, as the part that can use biotin derivative or the like is arranged, but said part is not limited to these as certification mark.When using luminophore, as the luminophore that can use acridinium ester, luminous element (luciferin), luminous plain enzyme (luciferase) etc. are arranged, but said luminophore is not limited to these as certification mark.When using nanoparticle, as the nanoparticle that can use Radioactive colloidal gold, colored latex (tinted latex) or the like are arranged, but said nanoparticle is not limited to these as certification mark.When using the redox molecule as certification mark; As the redox molecule that can use ferrocene, ruthenium complex, viologen (viologen), quinone, Ti ion, Cs ion, imide, 1 are arranged; 4-benzoquinones, quinhydrones, K4W (CN) 8, [Os (bpy) 3] 2+, [RU (bpy) 3] 2+, [MO (CN) 8] 4-etc., but said redox molecule is not limited to these.When using ri as certification mark; As the ri that can use 3H, 14C, 32P, 35S, 36Cl, 51Cr, 57Co, 58Co, 59Fe, 90Y, 125I, 131I or 186Re or the like are arranged, but said ri is not limited to these.
In aspect the 3rd of the present invention; There is level in " can the coded proteic material that has level and/or have pattern of the said prognosis in hcc affinity tag of specific detection " and/or has that specificity is used for the coded proteic any material of said affinity tag in the analytical procedure of material of pattern detecting coded proteic of said prognosis in hcc affinity tag, might not be limited to antibody.The technical characterictic of the 3rd aspect of the present invention be with coded proteic of the said prognosis in hcc affinity tag in the biological sample of taking from liver cancer patient have level or have pattern and benchmark relatively, and detect difference.Thereby; Any material all can be used as " can the coded proteic material that has level and/or have pattern of specific detection prognosis in hcc affinity tag " and can realize the technique effect that the present invention is intended to reach, as long as this material can detect such difference.Those of ordinary skill in the art can select/choose suitable material based on the general general knowledge and the known technology of this area according to embodiment easily.
The 4th aspect of the present invention relates to the test kit of predicting liver cancer prognosis, and said test kit includes the present invention second or the described compsn that is used for the predicting liver cancer prognosis of the third aspect.
Except be included in the compsn that is used for assessing prognosis in hcc can specific detection prognosis in hcc affinity tag expression level and/or the material of expression pattern; Or can the coded proteic material that has level and/or have pattern of specific detection prognosis in hcc affinity tag, the test kit that is used to assess prognosis in hcc among the present invention can also comprise there is level or has one or more other compositions, solution or the device of the method for pattern of method or analyzing proteins of the expression level that is applicable to analyzing gene or phraseology.For example; For the diagnostic kit that detects expression of gene level or expression pattern; This diagnostic kit can comprise and carries out the required fundamental component of RT-PCR; Except the specific separately primer of the mRNA of marker gene, this RT-PCR test kit according to concrete embodiment can comprise test tube for example or other containers that is fit to, reaction buffer, deoxynucleotide (dNTPs), enzyme for example Taq polysaccharase and reversed transcriptive enzyme, DNA enzyme, RNA enzyme inhibitors, DEPC-water (DEPC water), sterilized water, as the gene-specific primer equity of quantitative control group.Simultaneously, be used to detect at diagnostic kit and proteicly have level or exist under the situation of pattern, this diagnostic kit can comprise and for example carries out the required fundamental component of ELISA.This ELISA test kit can comprise the component that can detect binding antibody, for example, and two anti-, the chromophore of mark, enzyme (enzyme that for example is connected) and substrate thereof and to the special antibody of quantitative control group albumen with antibody.Further, according to concrete embodiment, this diagnostic kit can comprise dna microarray or arrays of immobilized protein.
The 5th aspect of the present invention relates to a kind of method that is used for prognosis in hcc assessment, and this method comprises: step 1, and use the compsn that is used to assess prognosis in hcc of second aspect to handle to taking from by the biological sample of examination liver cancer patient; With step 2, compare through result and benchmark step 1, test right requires the expression level of the prognosis in hcc affinity tag in 1 and/or the difference of expression pattern.In addition, the 5th aspect of invention relates to a kind of method that is used for prognosis in hcc assessment, and this method comprises: step 1, and use the compositions-treated that is used to assess prognosis in hcc of the third aspect to take from the biological sample of liver cancer patient; With step 2, compare through result and benchmark step 1, test right requires the coded albumen of the prognosis in hcc affinity tag in 1 having level and/or having the difference of pattern.
For example; Carry out expression level difference when detecting in the liver cancer tissue of prognosis in hcc good patient, expressing more prognosis in hcc affinity tag; If, can learn that the prognosis of liver cancer is better relatively from being higher than benchmark by the expression level of this affinity tag in the biological specimen of examination liver cancer patient.Simultaneously; For example carry out expression level difference when detecting in the patient's of prognosis in hcc difference liver cancer tissue, expressing more prognosis in hcc affinity tag; If, can learn that the prognosis of liver cancer is relatively poor relatively from being higher than benchmark by the expression level of this affinity tag in the biological specimen of examination liver cancer patient.
Simultaneously; For example; To in the liver cancer tissue of prognosis in hcc good patient, expressing the coded albumen of more prognosis in hcc affinity tag when existing level difference to detect; If, can learn that the prognosis of liver cancer is better relatively from being higher than benchmark by the coded proteic level that exists of this affinity tag in the biological specimen of examination liver cancer patient.Simultaneously; When for example the coded albumen of the more prognosis in hcc affinity tag of expression exists level difference to detect in the patient's relatively poor to prognosis in hcc liver cancer tissue; If, can learn that the prognosis of liver cancer is relatively poor relatively from being higher than benchmark by the coded proteic level that exists of this affinity tag in the biological specimen of examination liver cancer patient.
The 6th aspect of the present invention relates to the method that the prognosis in hcc affinity tag that uses first aspect of the present invention screens therapeutic agent for liver cancer.Because the prognosis in hcc affinity tag of first aspect of the present invention demonstrates the significant difference of expression pattern according to the prognosis of liver cancer patient, it can be to participate in directly with the generation of liver cancer or develop the gene of the relevant physiologic function of particularly prognosis.Therefore, the coded albumen of this affinity tag can be effectively with the generation of the liver cancer that makes a search or the target protein of development mechanism or invention therapeutic agent for liver cancer.Therefore that is, the said prognosis in hcc affinity tag in first aspect of the present invention meets the important prerequisite of the therapeutical agent of developing liver cancer, and this affinity tag of this use method of screening therapeutic agent for liver cancer also is encompassed in protection scope of the present invention.
As an instance of the 6th aspect of the present invention, the method for the therapeutical agent of screening liver cancer is provided, said method comprises and detects the step of expression whether test compounds can promote or suppress the prognosis in hcc affinity tag of first aspect of the present invention.As the method for screening therapeutical agent, can use for example RT-PCR, competitive RT-PCR, real-time RT-PCR, the experiment of RNA enzyme protection, the northern marking, dna microarray, SAGE [serial analysis of gene expression; Velculescu etc., Science 270:484-7], MPSS [extensive parallel mark order-checking; Brenner etc., PNAS.USA.97,1665-1670] etc.Except above method, can use the various known method of this area if necessary.
Simultaneously, as another instance of the 6th aspect of the present invention, the method for the therapeutical agent of screening liver cancer is provided, said method comprises: step 1, the protein binding that the prognosis in hcc affinity tag of test compounds and first aspect of the present invention is coded; With step 2, detect this test compounds and can promote or suppress said proteic physiologically active.As the method for screening therapeutical agent, for example, can use the protein marker that is used for hepatocarcinoma early diagnosis to be fixed to the method [Pandya etc. that contact purification of samples on the affinity column and with itself and sample with first aspect of the present invention; Virus Res 87:135-143; 2002], use the method for yeast two-hybrid system, western blotting, the method [Aviezer etc. of high flux screening; J Biomol Screen 6:171-7,2001] etc.Except above method, can use the various known method of this area if necessary.
In aspect the 6th of the present invention; As the test compounds that is used to screen; Can use the expression product, synthetic compound, synthetic polypeptide, natural compounds of for example tissue extract, gene library etc., but the test compounds that is used to screen is not limited to these.
The 7th aspect of the present invention relates to the antibody of specific recognition by the prognosis in hcc affinity tag encoded protein of first aspect of the present invention.
The antibody of the prognosis in hcc affinity tag of specific recognition first aspect of the present invention is the coded proteic representative substances that has level or have pattern of specific detection prognosis in hcc affinity tag, thereby can be used to assess prognosis in hcc effectively.In addition, according to circumstances, said antibody can specificity promotes or is suppressed at the proteic activity that plays an important role in generation or the development of liver cancer, thereby can be with the therapeutical agent that acts on liver cancer.
Owing to found the prognosis in hcc affinity tag of first aspect of the present invention, can easily carry out the preparation of coded proteic polyclonal antibody, monoclonal antibody and recombinant antibodies of said prognosis in hcc affinity tag through the technology of using this area likewise known.
In the coded protein injection precession object of the prognosis in hcc affinity tag with first aspect of the present invention of this area likewise known, and, can prepare polyclonal antibody from animal blood sampling method with the serum that obtains to comprise antibody.These polyclonal antibodies can prepare from various animal hosts, for example goat, rabbit, sheep, monkey, horse, pig, ox, dog etc., and the preparation method is well known in the art.
Monoclonal antibody can be through using hybridoma method [Kohler and Milstein (1976) European Jounral of Immunology 6:511-519] or phage antibody library technology [Clackson etc., Nature, 352:624-628,1991; Marks etc., J.Mol.Biol., 222:58,1-597,1991] etc. the method for this area likewise known prepare.Usually; Hybridoma method is used the for example cell that obtains of mouse and as the cancer or the myeloma cell line of another colony of the host animal that is fit to from immunology, and said animal has been injected the coded proteantigen of prognosis in hcc affinity tag of first aspect of the present invention.The tissue that from two colonies, is obtained merges through the method like this area likewise known such as polyoxyethylene glycol, makes antibody-producting cell propagation with the normal structure cultural method then.After obtaining the homogenous cell crowd according to limiting dilution technique through subclone, according to known technique in vivo or the external hybridoma that can produce required antibody of cultivating in large quantities.The phage antibody library method is a kind of like this preparation monoclonal antibody method: the gene that obtains required antibody; On phage surface with this gene of formal representation of fusion rotein; Thereby, and from this library, separate monoclonal antibody at external generation antibody library.Monoclonal antibody through above method preparation can be separated through using currently known methodss such as example gel electrophoresis, dialysis, salt deposition, ion exchange chromatography, affinity chromatography.
The antibody of the 7th aspect of the present invention also comprises the function fragment of antibody molecule except the perfect shape that comprises two full-length light chains and two total length heavy chains.The function fragment of said antibody molecule is meant the fragment with antigen combined function, comprises Fab, F (ab '), F (ab ') 2, Fv or the like.
The invention effect
According to the present invention, a kind of prognosis in hcc affinity tag is provided; A kind of compsn that is used to assess prognosis in hcc, said composition comprises the material of the changes of expression level that can detect said prognosis in hcc affinity tag; A kind of test kit that is used to assess prognosis in hcc, said test kit comprise the said compsn that is used to assess prognosis in hcc; A kind of method of using said prognosis in hcc affinity tag to carry out the prognosis in hcc assessment; And a kind of method of using said prognosis in hcc affinity tag screening therapeutic agent for liver cancer.
Said prognosis in hcc affinity tag can be used for the simple and correct prognosis evaluation of liver cancer patient effectively.In addition, the physiologic function of said affinity tag can be directly with the generation of liver cancer or develop relevant.Thereby this affinity tag can be effective to the generation of liver cancer or the research of development mechanism, or as the target of developing therapeutic agent for liver cancer.
Above-mentioned prognosis in hcc affinity tag is the useful and affinity tags of predicting liver cancer prognosis evaluation more accurately of those more significant, clinical height, and this is because they are to find through the liver cancer tissue of taking from unprecedented multidigit patient is carried out statistical analysis.
Description of drawings
Fig. 1 to Figure 10 is through measuring said prognosis in hcc affinity tag at the Kaplan-Meier curve that illustrates aspect recurrence, existence always and the DFS.
Embodiment
Can set forth the present invention in detail through embodiment below; Yet, describe said embodiment and only be in order to help to understand the present invention, rather than limit scope of the present invention by any way.
Embodiment
The extraction of embodiment 1:RNA and cDNA's is synthetic
Acquisition is taken from 120 and has been diagnosed as that liver cancer has taken place and the liver cancer tissue of the liver cancer patient that the development of liver cancer has obtained confirming and adjacent healthy tissues.Extract the RNA of each part tissue and carry out the synthetic of cDNA according to following method.
According to product description, use RNeasy Minikit (Qiagen, Germany) from liver cancer tissue and adjacent healthy tissues, to extract total RNA.Use Bioanalyzer 2100 (Agilent Technologies, the U.S.) to the total RNA weighing in the RNA extract that obtains.The processing of in the step of extracting, carrying out DNase I is to remove the genomic dna that depollutes from the RNA extract.(Genotech is Korea) 70 ℃ of incubations 7 minutes and cooled on ice 5 minutes to contain d (T) 18 primers of 1 μ M of sample and 2 μ l of the total RNA of 4 μ g.The preparation enzyme mixture is [through adding the DTT (Duchefa of 2 μ l 0.1M separately; Netherlands), the 40U/ μ l RNA enzyme inhibitors of the MMLV reversed transcriptive enzyme of the dNTP of 10 * rt damping fluid of 2 μ l, 5 μ l 2mM, 1 μ l 200U/ μ l and 1 μ l (Enzynomics, Korea) to TV be 11 μ l].After being added into this enzyme mixture in the sample that contains RNA, it 42 ℃ of incubations 90 minutes, was made the reversed transcriptive enzyme inactivation in 10 minutes at 80 ℃ of incubations then.Making the final volume of above-mentioned sample through the water that adds diethylpyrocarbonate (DEPC) processing is 400 μ l.
Embodiment 2: quantitatively real-time RCR
According to product manual, use PRISM 7900HT (Applied Biosystems, the U.S.) that following two gene markers of each cDNA sample of acquisition among the embodiment 1 are implemented quantitatively RCR amplification in real time:
CBS (cystathionine beta-synthase; NCBI GI:209862802; SEQ ID NO:79); With
NNMT (NNMT; NCBI GI:62953139; SEQ ID NO:80).
Said quantitatively RCR analysis is in real time carried out in the TV of 10 μ l; Said TV comprises 2 * Taqman genetic expression master primer (master mix) (Applied Biosystems of 5 μ l; USA), (Genotech is Korea) with the cDNA (being the water of equivalent in control group) of 2 μ l for the 1 μ M probe of each 1 μ l of 5 μ M forwards and reverse primer, 1 μ l.Amplification is carried out with following circulation: 95 ℃ of steps of dissociating 10 minutes are 95 ℃ of steps of dissociating 15 seconds and 60 ℃ of steps of synthetic 1 minute then.Primer and probe sequence use the design of Primer Express 3.0 (Applied Biosystems, the U.S.), and all probe sequences indicate FAM at 5 ' end, and 3 ' end indicates TAMRA.Below following primer and probe in the table 1 be used for every kind of affinity tag.
Table 1
Figure BDA0000108634200000151
F: forward primer
R: reverse primer
P: probe
The mensuration of every kind of marker gene expression is triplicate all, comes stdn through subtracting each other with 5 kinds of average expression with reference to gene (B2M, GAPDH, HMBS, HPRT1 and SDHA) then.Measure the CT (reaching the required cycle number of threshold value) of every kind of affinity tag, and calculate Δ CT value (CT of every kind of affinity tag deducts the average CT of said reference gene).According to 2 -Δ CTCalculate the copy number of mRNA.Amplification makes up typical curve in the time of according to the cDNA sample after a series of dilutions.
Embodiment 3: data analysis
Consider among the embodiment 2 the standardized expression of the every kind of affinity tag that obtains and the patient's of liver cancer tissue progress is provided, accomplished the Kaplan-Meier curve, carry out significance analysis subsequently.
According to 120 progress that the patient of liver cancer tissue is provided, for the case separately of recurrence, existence always and DFS, said patient lists with the order of rising progressively period.Through be exposed to patient's quantity in the risk remove existent's (or patient of recurrence) quantity calculate in the middle of survival rate (interval survival rate) (or middle recurrence rate (interval recurrence rate)).Accumulation survival rate (or cumulative relapse frequencies) is a conditional probability, and it multiply by present middle recurrence rate (middle recurrence rate) through previous accumulation survival rate (or cumulative relapse frequencies) and calculates.With survival time (or observation period) be transverse axis and with the accumulation survival rate (or cumulative relapse frequencies) be the longitudinal axis, make up the Kaplan-Meier curve through step function.
For every kind of affinity tag, accomplished Kaplan-Meier curve about recurrence, existence always and DFS.Be divided into high expression level and low expression according to testing of the expression of definite remarkable benchmark of statistics, and, high expression level situation and low expression be separated from each other for each affinity tag with each affinity tag of measuring among the embodiment 2, thus completion Kaplan-Meier curve.Fig. 1 shows the Kaplan-Meier curve of having accomplished.
Can confirm that by Fig. 1 in the Kaplan-Meier curve of accomplishing for recurrence, existence always and DFS, every kind of affinity tag has formed the curve that high expression level situation and low expression are obviously distinguished each other.This means at each affinity tag and show as between high expression level and the low situation about expressing; There are evident difference in middle recurrence rate or middle survival rate and cumulative relapse frequencies based on this or accumulation survival rate; And mean that therefore the expression pattern of every kind of affinity tag can be to show that the patient is recurred possibility or the index of the possibility of surviving.
Observed value and desired value through calculating each recurrence point or death point obtain the chi square test Statistical information, and each affinity tag or its combination are carried out test of significance through log rank test (log-rank test).Thereby calculate the p value, following table 2 has shown the p value that calculates.
Table 2
Figure BDA0000108634200000161
Can confirm that from above-mentioned table 2 each affinity tag or its combination have shown enough low p value, to such an extent as to can think all have significance for recurrence, existence always and DFS.Particularly, under the situation of two kinds of affinity tag combinations, all p values of recurrence, existence always and DFS are all less than 0.05, and this is desirable.When the p value becomes low more, it is high more that significance,statistical just becomes.Therefore, low p value means that through each affinity tag or its assessment of making up prognosis in hcc be highly accurate.
Embodiment 4: the discovery of other affinity tag
According to testing with embodiment 1 to embodiment 3 identical mode; Difference is to experimentize with the cancerous tissue of taking from 185 liver cancer patients and adjacent healthy tissues, thereby through using the following gene thing that serves as a mark to obtain Kaplan-Meier curve and p value:
TKT (transketolase; NCBI GI:205277461; SEQ ID NO:81).
Following table 3 has shown employed primer and probe, and Fig. 2 has shown the Kaplan-Meier curve; And following table 4 has shown the p value that calculates.
Table 3
Figure BDA0000108634200000171
F: forward primer
R: reverse primer
P: probe
Table 4
Figure BDA0000108634200000172
Can find out that by Fig. 2 in the Kaplan-Meier curve that recurrence, existence always and DFS are accomplished, above-mentioned affinity tag has formed high expression level situation and the remarkable each other curve of distinguishing of low expression.This means between this affinity tag high expression level and low situation about expressing; Middle recurrence rate or middle survival rate; There are significant difference in cumulative relapse frequencies based on this or accumulation survival rate; And mean that therefore the expression pattern of this affinity tag can be to show that the patient is recurred possibility or the index of the possibility of surviving.
Can confirm that from above-mentioned table 4 aspect recurrence, existence always and all of DFS, above-mentioned affinity tag demonstrates the p value less than 0.05, this is desirable low.When the p value became lower, it is higher that significance,statistical becomes.Therefore, low p value means that be highly accurate through this affinity tag to the assessment of prognosis in hcc.
Embodiment 5: the discovery of other affinity tag
According to testing with embodiment 1 to embodiment 3 identical mode; Difference is to experimentize with the cancerous tissue of taking from 369 liver cancer patients and adjacent healthy tissues, thereby utilizes following 23 genes thing that serves as a mark to obtain Kaplan-Meier curve and p value:
AIFM1 (apoptosis inducing factor, plastosome; NCBI GI:22202627; SEQ ID NO:82);
AKT1 (RAC-α serine/threonine protein kitase; NCBI GI:62241010; SEQ ID NO:83);
ATG3 (autophagy GAP-associated protein GAP 3; NCBI GI:34147490; SEQ ID NO:84);
ATG5 (autophagy GAP-associated protein GAP 5; NCBI GI:92859692; SEQ ID NO:85);
ATG7 (autophagy GAP-associated protein GAP 7; NCBI GI:222144225; SEQ ID NO:86);
ATG12 (autophagy correlated protein 12; NCBI GI:38261968; SEQ ID NO:87);
BAX (apoptosis regulatory factor BAX; NCBI GI:34335114; SEQ ID NO:88);
BCL2 (apoptosis regulatory factor Bcl-2; NCBI GI:72198188; SEQ ID NO:89);
BCL2L1 (apoptosis regulatory factor Bcl-X; NCBI GI:20336333; SEQ ID NO:90);
BNIP3 (BCL2/ adenovirus E 1 B 19kD albumen-interaction protein 3; NCBI GI:7669480; SEQ ID NO:91);
CASP8 (caspase 8; NCBI GI:122056470; SEQ ID NO:92);
CSE1L(Exportin-2；NCBI?GI：29029558；SEQ?ID?NO：93)；
DIABLO (Diablo homologue, plastosome; NCBI GI:218505810; SEQ ID NO:94);
(the autophagy modulin is regulated in damage to DRAM; NCBI GI:110825977; SEQ ID NO:95);
E2F1 (transcription factor E2F1; NCBI GI:168480109; SEQ ID NO:96);
FAS (tumor necrosis factor receptor super family member 6; NCBI GI:23510419; SEQ ID NO:97);
FRAP1 (FKBP12-Wyeth-Ayerst Laboratories compound relative protein; NCBI GI:206725550; SEQ ID NO:98);
LAMP1 (lysosome related film gp 1; NCBI GI:112380627; SEQ ID NO:99);
LC3 [MAP1LC3A] (microtubule bindin 1A/1B light chain 3A; NCBI GI:31563519; SEQ ID NO:100);
PRKAA1 (5 '-AMP activated protein kinase catalytic subunit α-1; NCBI GI:94557300; SEQ ID NO:101);
PTEN (PI-3,4, the phosphoprotein phosphatase PTEN of 5-triphosphoric acid 3-Phosphoric acid esterase and dual specific; NCBI GI:110224474; SEQ ID NO:102);
ULK1 (serine/threonine protein kitase ULK1; NCBI GI:225637564; SEQ ID NO:103); With
XIAP (the albumen 4 that contains baculovirus IAP Tumor-necrosis factor glycoproteins; NCBI GI:32528298; SEQ ID NO:104).
Following table 5 has shown employed primer and probe, and Fig. 3 to Figure 10 has shown the Kaplan-Meier curve; Following table 6 has shown the p value that calculates for each affinity tag, and following table 7 to table 9 has shown the p value that combination calculated for any two kinds of affinity tags.
Table 5
Figure BDA0000108634200000191
Table 6
Figure BDA0000108634200000201
Table 7
Table 8
Table 9
Can confirm that by Fig. 3 to Figure 10 in the Kaplan-Meier curve of accomplishing for recurrence, existence always and DFS, each affinity tag has formed high expression level situation and the remarkable each other curve of distinguishing of low expression.This means at each affinity tag and show as between high expression level and the low situation about expressing; Middle recurrence rate or middle survival rate; There are evident difference in cumulative relapse frequencies based on this or accumulation survival rate; And therefore, the expression pattern of each affinity tag can be to show that the patient is recurred possibility or the index of the possibility of surviving.
Can confirm that from above-mentioned table 6 to table 9 combination of every kind of affinity tag or all two kinds of affinity tags has shown enough low p value, to such an extent as to can think that it has significance aspect recurrence, existence always and the DFS.When the p value became lower, it is higher that significance,statistical becomes.Therefore, low p value means that the assessment of the prognosis in hcc that carries out through every kind of affinity tag or their combination is accurately.Particularly, most each affinity tag or two kinds of all affinity tag combinations all demonstrate the p value less than 0.05, and this is desirable low p value.
The p value of all combinations of affinity tag more than three kinds is all less than 0.05.Below table 10 provided the combination of the single labelled thing that demonstrates minimum p value or two or more affinity tag and p value accordingly.
Table 10
[recurrence :]
Figure BDA0000108634200000241
Figure BDA0000108634200000243
Can find out through above table 10, can find that the p value is just low more along with the affinity tag of the present invention of combination is many more.This means the affinity tag of the present invention that combination is more, demonstrate low more p value (the low more significance that means of p value is high more) for a long time, thereby show the accuracy that has realized further improvement in the prognosis evaluation based on the affinity tag combination.
Embodiment 6: cross validation
Cross validation has been carried out in combination to the affinity tag that in embodiment 5, has significance,statistical.
369 liver cancer patients are divided into two groups of (positive groups: 185 patients at random; Test group: 184 patients).For positive group, according to embodiment 3 in identical method set up through experiment and be considered to significant benchmark on the statistics, and carried out high expression level and the low classification of expressing.Benchmark through setting up is like this fixed, for test group, calculate in p＜0.05 or p＜0.001 o'clock to the levels of accuracy of the assessment of recurrence, existence always and DFS.
Be the representative example that the prognosis of recurrence, existence always and DFS is shown excellent particularity below:
Recurrence: AIFM1_AKT1_LC3 (77.3%, the level of p＜0.05)
Total existence: ATG5_DRAM_FAS_XIAP (87.3%, the level of p＜0.001)
DFS: AIFM1_AKT1_LC3 (71.3%, the level of p＜0.05).
Figure IDA0000108634270000011
Figure IDA0000108634270000021
Figure IDA0000108634270000031
Figure IDA0000108634270000041
Figure IDA0000108634270000051
Figure IDA0000108634270000061
Figure IDA0000108634270000071
Figure IDA0000108634270000081
Figure IDA0000108634270000091
Figure IDA0000108634270000111
Figure IDA0000108634270000121
Figure IDA0000108634270000131

Claims (11)

1. prognosis in hcc affinity tag, said affinity tag comprise and being selected from by a gene of the group of following genomic constitution or the combination of at least two genes:
CBS (cystathionine beta-synthase; NCBI GI:209862802; SEQ ID NO:79);
NNMT (NNMT; NCBI GI:62953139; SEQ ID NO:80);
TKT (transketolase; NCBI GI:205277461; SEQ ID NO:81);
AIFM1 (apoptosis inducing factor 1, plastosome; NCBI GI:22202627; SEQ ID NO:82);
AKT1 (RAC-α serine/threonine protein kitase; NCBI GI:62241010; SEQ ID NO:83);
ATG3 (autophagy GAP-associated protein GAP 3; NCBI GI:34147490; SEQ ID NO:84);
ATG5 (autophagy GAP-associated protein GAP 5; NCBI GI:92859692; SEQ ID NO:85);
ATG7 (autophagy GAP-associated protein GAP 7; NCBI GI:222144225; SEQ ID NO:86);
ATG12 (autophagy correlated protein 12; NCBI GI:38261968; SEQ ID NO:87);
BAX (apoptosis regulatory factor BAX; NCBI GI:34335114; SEQ ID NO:88);
BCL2 (apoptosis regulatory factor Bcl-2; NCBI GI:72198188; SEQ ID NO:89);
BCL2L1 (apoptosis regulatory factor Bcl-X; NCBI GI:20336333; SEQ ID NO:90);
BNIP3 (BCL2/ adenovirus E 1 B 19kDa albumen-interaction protein 3; NCBI GI:7669480; SEQ ID NO:91);
CASP8 (caspase 8; NCBI GI:122056470; SEQ ID NO:92);
CSE1L(Exportin-2；NCBI?GI：29029558；SEQ?ID?NO：93)；
DIABLO (Diablo homologue, plastosome; NCBI GI:218505810; SEQ ID NO:94);
(the autophagy modulin is regulated in damage to DRAM; NCBI GI:110825977; SEQ ID NO:95);
E2F1 (transcription factor E2F1; NCBI GI:168480109; SEQ ID NO:96);
FAS (tumor necrosis factor receptor super family member 6; NCBI GI:23510419; SEQ ID NO:97);
FRAP1 (FKBP12-Wyeth-Ayerst Laboratories compound relative protein; NCBI GI:206725550; SEQ ID NO:98);
LAMP1 (lysosome related film gp 1; NCBI GI:112380627; SEQ ID NO:99);
LC3 [MAP1LC3A] (microtubule bindin 1A/1B light chain 3A; NCBI GI:31563519; SEQ ID NO:100);
PRKAA1 (5 '-AMP activated protein kinase catalytic subunit α-1; NCBI GI:94557300; SEQ ID NO:101);
PTEN (PI-3,4, the phosphoprotein phosphatase PTEN of 5-triphosphoric acid 3-Phosphoric acid esterase and dual specific; NCBI GI:110224474; SEQ ID NO:102);
ULK1 (serine/threonine protein kitase ULK1; NCBI GI:225637564; SEQ ID NO:103); With
XIAP (the albumen 4 that contains baculovirus IAP Tumor-necrosis factor glycoproteins; NCBI GI:32528298; SEQ ID NO:104).
2. compsn that is used to assess prognosis in hcc, said compsn comprise the expression level that is used for the described prognosis in hcc affinity tag of specific detection claim 1 and/or the material of expression pattern.
3. compsn that is used to assess prognosis in hcc, said compsn comprises and is used for the coded proteic material that has level and/or have pattern of the described prognosis in hcc affinity tag of specific detection claim 1.
4. the compsn that is used to assess prognosis in hcc as claimed in claim 2; Wherein, the material of said expression level that is used for the said prognosis in hcc affinity tag of specific detection and/or expression pattern is the used primer of RT-PCR that detects the mRNA of said prognostic markers thing.
5. the compsn that is used to assess prognosis in hcc as claimed in claim 3, wherein, said to be used for that there is level in coded proteic of the said prognosis in hcc affinity tag of specific detection and/or has the material of pattern be the said proteic antibody of specific recognition.
6. test kit that is used to assess prognosis in hcc, said test kit comprises each described compsn that is used to assess prognosis in hcc in the claim 2 and 5.
7. method of assessing prognosis in hcc, said method comprises:
Step 1 is taken from by the biological sample of examination liver cancer patient with claim 2 or the 4 described compositions-treated that are used to assess prognosis in hcc; With
Step 2, thereby the difference that requires the expression level and/or the expression pattern of 1 described prognosis in hcc affinity tag through result and benchmark comparison test right with step 1.
8. method of assessing prognosis in hcc, said method comprises:
Step 1 is taken from the biological sample of liver cancer patient with claim 3 or the 5 described compositions-treated that are used to assess prognosis in hcc; With
Step 2, thus the coded proteic difference that has level and/or have pattern of 1 described prognosis in hcc affinity tag required through result and benchmark comparison test right with step 1.
9. method of screening therapeutic agent for liver cancer, said method comprise and detect the step whether test compounds promotes or suppress the expression of the described prognosis in hcc affinity tag of claim 1.
10. method of screening therapeutic agent for liver cancer, said method comprises:
Step 1 is with test compounds and the coded protein binding of the described prognosis in hcc affinity tag of claim 1; With
Whether step 2 detects said test compounds and promotes or suppress said proteic physiologically active.
11. coded proteic antibody of the described prognosis in hcc affinity tag of specific recognition claim 1.
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